Challenges in RNA virus bioinformatics

Motivation: Computer-assisted studies of structure, function and evolution of viruses remains a neglected area of research. The attention of bioinformaticians to this interesting and challenging field is far from commensurate with its medical and biotechnological importance. It is telling that out of >200 talks held at ISMB 2013, the largest international bioinformatics conference, only one presentation explicitly dealt with viruses. In contrast to many broad, established and well-organized bioinformatics communities (e.g. structural genomics, ontologies, next-generation sequencing, expression analysis), research groups focusing on viruses can probably be counted on the fingers of two hands. Results: The purpose of this review is to increase awareness among bioinformatics researchers about the pressing needs and unsolved problems of computational virology. We focus primarily on RNA viruses that pose problems to many standard bioinformatics analyses owing to their compact genome organization, fast mutation rate and low evolutionary conservation. We provide an overview of tools and algorithms for handling viral sequencing data, detecting functionally important RNA structures, classifying viral proteins into families and investigating the origin and evolution of viruses. Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online. The references for this article can be found in the Supplementary Materia

Analysis of NGS Data from Immune Response and Viral Samples

This thesis is devoted to designing and applying advanced algorithmical and statistical tools for analysis of NGS data related to cancer and infection diseases. NGS data under investigation are obtained either from host samples or viral variants. Recently, random peptide phage display libraries (RPPDL) were applied to studies of host\u27s antibody response to different diseases. We study human antibody response to breast cancer and mouse antibody response to Lyme disease by sequencing of the whole antibody repertoire profiles which are represented by RPPDL. Alternatively, instead of sequencing immune response NGS can be applied directly to a viral population within an infected host. Specifically, we analyze the following RNA viruses: the human immunodeficiency virus (HIV) and the infectious bronchitis virus (IBV). Sequencing of RNA viruses is challenging because there are many variants inside population due to high mutation rate. Our results show that NGS helps to understand RNA viruses and explore their interaction with infected hosts. NGS also helps to analyze immune response to different diseases, trace changing of immune response at different disease stages

The International Virus Bioinformatics Meeting 2020.

The International Virus Bioinformatics Meeting 2020 was originally planned to take place in Bern, Switzerland, in March 2020. However, the COVID-19 pandemic put a spoke in the wheel of almost all conferences to be held in 2020. After moving the conference to 8-9 October 2020, we got hit by the second wave and finally decided at short notice to go fully online. On the other hand, the pandemic has made us even more aware of the importance of accelerating research in viral bioinformatics. Advances in bioinformatics have led to improved approaches to investigate viral infections and outbreaks. The International Virus Bioinformatics Meeting 2020 has attracted approximately 120 experts in virology and bioinformatics from all over the world to join the two-day virtual meeting. Despite concerns being raised that virtual meetings lack possibilities for face-to-face discussion, the participants from this small community created a highly interactive scientific environment, engaging in lively and inspiring discussions and suggesting new research directions and questions. The meeting featured five invited and twelve contributed talks, on the four main topics: (1) proteome and RNAome of RNA viruses, (2) viral metagenomics and ecology, (3) virus evolution and classification and (4) viral infections and immunology. Further, the meeting featured 20 oral poster presentations, all of which focused on specific areas of virus bioinformatics. This report summarizes the main research findings and highlights presented at the meeting

Understanding the Molecular Basis of Disease Resistance Against Septoria Tritici Blotch in Wheat

Zymoseptoria tritici, a fungal pathogen of wheat and the causal agent of Septoria Tritici Blotch (STB), is a particular challenge to wheat production on a global level, causing crop losses of up to 50% in some cases. Genetic resistance presents an effective way of controlling STB in wheat, with numerous resistance loci having been identified. Stb6, which encodes a wall-associated kinase-like protein and confers resistance to Z. tritici isolates expressing the corresponding AvrStb6 effector, has recently been cloned. However, many questions regarding Stb6-mediated resistance remain unanswered. The global frequency of avirulence AvrStb6 alleles in modern Z. tritici populations remains unknown. The role that AvrStb6 plays in Z. tritici pathogenicity, the nature of its interaction with Stb6 and the mechanism by which Stb6 confers resistance are also yet to be elucidated. In the following thesis, I present data indicating a dramatic shift in the frequency of virulence AvrStb6 isoforms in modern Z. tritici populations, relative to previous studies. Transient expression of fluorophore-tagged Stb6 and AvrStb6 in the tobacco species Nicotiana benthamiana also revealed that AvrStb6 localises to the apoplast. Stb6 displays an unusual cellular localisation pattern, being present in both the plasma membrane but also identified in the cell wall and in hectian strands adjoining the two. Expression of Stb6 in N. benthamiana induces a kinase-dependent cell death phenotype, which is independent of the co-receptor BAK1. I also report on the identification of candidate protein interactors for Stb6 and AvrStb6. Both interactors are predicted to be chloroplast-localised. These findings present an intriguing hypothesis as to the possible mechanism of Stb6-mediated resistance. This study therefore represents an important contribution to our understanding of evolutionary genetics in a plant pathogen, provides a putative mechanism for AvrStb6 in pathogenicity and has also potentially identified a completely novel resistance mechanism for an immune receptor protein. Research such as this plays a role in helping develop more productive crop plants with improved genetic resistance to pathogens

Transmission Dynamics and Epidemiology of West Nile Virus in Ontario, Canada

The resurgence of West Nile virus (WNV; Family Flaviviridae, genus Flavivirus) in Ontario, Canada in 2012 demonstrated that there is a great need for a reassessment of the local mosquito fauna, estimation of risk of WNV transmission, and the creation of effective arboviral awareness campaigns. A review of the current literature and collection databases revealed that there are 68 mosquito species known from Ontario (Chapter 2). Ten species were added to the list of species including Culex erraticus (Chapter 3) and Aedes albopictus (Chapter 4), both of which are capable of transmitting West Nile virus. Ae. albopictus was repeatedly collected from Windsor, Ontario in 2016 (Chapter 4). Immatures (n=78) were collected from tires, StyrofoamTM containers, and discarded garbage. Adult female (n=17) and male (n=2) specimens were collected from light traps (n=7) and Biogents-Sentinel traps (n=10). Additional specimens were obtained from Franklin County, Ohio. The generated gene tree and Bayesian cluster analysis grouped sequences described from Ohio and Windsor together on the same branches. Together these data suggest that the population in Windsor originated as a founder population of North American origin by means of human-aided dispersal. Mosquito abundance predication surfaces and seasonal distributions were attempted for each vector species to identify where and when vector species are most abundant in southern Ontario (Chapter 5). Spatial prediction surfaces using kriging were created for Aedes vexans, Aedes japonicus, Culex pipiens, Culex restuans, and Ochlerotatus trivittatus. Proximity to landscape variables was observed to improve model prediction. An epidemiological analysis of WNV human case prevalence and mosquito infection was conducted (Chapter 6). A strong quadratic relationship between the number of human cases and positive mosquito pools at the end of each year was observed (R2=0.9783, p < 0.001). Spearman rank correlation tests identified mosquito infection rates as the strongest predictors of human case prevalence at a one-week lag period. Average temperature was a strong predictor of mosquito infection rates. Cumulative positive Culex pools recorded by epidemiological week 34 is a sufficient action threshold for West Nile virus epidemics. These data have the potential to contribute to a more efficient West Nile virus awareness campaign

The International Virus Bioinformatics Meeting 2020.

The International Virus Bioinformatics Meeting 2020 was originally planned to take place in Bern, Switzerland, in March 2020. However, the COVID-19 pandemic put a spoke in the wheel of almost all conferences to be held in 2020. After moving the conference to 8-9 October 2020, we got hit by the second wave and finally decided at short notice to go fully online. On the other hand, the pandemic has made us even more aware of the importance of accelerating research in viral bioinformatics. Advances in bioinformatics have led to improved approaches to investigate viral infections and outbreaks. The International Virus Bioinformatics Meeting 2020 has attracted approximately 120 experts in virology and bioinformatics from all over the world to join the two-day virtual meeting. Despite concerns being raised that virtual meetings lack possibilities for face-to-face discussion, the participants from this small community created a highly interactive scientific environment, engaging in lively and inspiring discussions and suggesting new research directions and questions. The meeting featured five invited and twelve contributed talks, on the four main topics: (1) proteome and RNAome of RNA viruses, (2) viral metagenomics and ecology, (3) virus evolution and classification and (4) viral infections and immunology. Further, the meeting featured 20 oral poster presentations, all of which focused on specific areas of virus bioinformatics. This report summarizes the main research findings and highlights presented at the meeting

Detecting Selection on Noncoding Nucleotide Variation: Methods and Applications

There has been a long tradition in molecular evolution to study selective pressures operating at the amino-acid level. But protein-coding variation is not the only level on which molecular adaptations occur, and it is not clear what roles non-coding variation has played in evolutionary history, since they have not yet been systematically explored. In this dissertation I systematically explore several aspects of selective pressures of noncoding nucleotide variation: The first project (Chapter 2) describes research on the determinants of eukaryotic translation dynamics, which include selection on non-coding aspects of DNA variation. Deep sequencing of ribosome-protected mRNA fragments and polysome gradients in various eukaryotic organisms have revealed an intriguing pattern: shorter mRNAs tend to have a greater overall density of ribosomes than longer mRNAs. There is debate about the cause of this trend. To resolve this open question, I systematically analysed 5’ mRNA structure and codon usage patterns in short versus long genes across 100 sequenced eukaryotic genomes. My results showed that compared with longer ones, short genes initiate faster, and also elongate faster. Thus the higher ribosome density in short eukaryote genes cannot be explained by translation elongation. Rather it is the translation initiation rate that sets the pace for eukaryotic protein translation. This work was followed by modelling studies of translation dynamics in a yeast cell. Chapter 3 concerns detecting selective pressures on the viral RNA structures. Most previous research on RNA viruses has focused on identifying amino-acid residues under positive or purifying selection, whereas selection on RNA structures has received less attention. I developed algorithms to scan along the viral genome and identify regions that exhibit signals of purifying or diversifying selection on RNA structure, by comparing the structural distances between actual viral RNA sequences against an appropriate null distribution. Unlike other algorithms that identify structural constraints, my approach accounts for the phylogenetic relationships among viral sequences, as well the observed variation in amino-acid sequences. Applied to Influenza viruses, I found that a significant portion of influenza viral genomes have experienced purifying selection for RNA structure, in both the positive- and negative-sense RNA forms, over the past few decades; and I found the first evidence of positive selection on RNA structure in specific regions of these viral genomes. Overall, the projects presented in these chapters represent a systematic look at several novel aspects of selection on noncoding nucleotide variation. These projects should open up new directions in studying the molecular signatures of natural selection, including studies on interactions between different layers at which selection may operate simultaneously (e.g. RNA structure and protein sequence)

The Evolution of the Viral RNA Sensor OAS1 in Old World Monkeys and Cetartiodactyls

Animals produce an array of sensors patrolling the intracellular environment poised to detect and respond to viral infection. The oligoadenylate synthetase family of enzymes comprises a crucial part of this innate immune response, directly signaling endonuclease activity responsible for inhibiting viral replication. Oligoadenylate synthetase 1 plays a vital role in animal susceptibility to pathogens including flaviviruses such as dengue, West Nile, and hepatitis c virus. This thesis includes a population level analysis of OAS1 diversity within macaque and baboon species followed by a broader survey of the gene in nineteen Old World monkeys. My research found that at the species level, macaques exhibit extremely high diversity with intriguing similarities to that previously found in chimpanzees. Across Old World monkeys, I identified commonly shared patterns of positive selection. Detailed structural analysis of OAS1 variation indicates sites of accelerated evolution at the host-virus interface and at sites of possible viral antagonism, both signifying a history of virus-driven evolution. Finally, I have analyzed the cetartiodactyl OAS1 gene family which includes two, and in some lineages, three copies of OAS1. This analysis identifies some of the same regions under evolutionary pressure as found in primates and also highlights others suggesting neofunctionalization of paralogous genes

A genomic analysis using RNA-Seq to investigate the adaptation of the psychrophilic diatom Fragilariopsis cylindrus to the polar environment

Diatoms are unicellular photosynthetic eukaryotes with a silicate cell wall. They often dominate polar marine ecosystems, driving the major biogeochemical cycles in these areas. The obligate psychrophilic diatom Fragilariopsis cylindrus is a keystone species in the Southern Ocean. It thrives both in open waters and sea ice and has become a model for studying eukaryotic microalgal adaptations to polar marine conditions. The aim of this thesis was to identify how the genome of F. cylindrus has evolved to cope with marine environmental conditions of the Southern Ocean. To identify key genes, comparative genomics, high-throughput transcriptome sequencing and reverse genetics were applied. Comparative genomics with the sequenced mesophilic diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana was combined with genome-wide RNA-Seq transcriptome analysis, leading to the discovery a new bacteria-like rhodopsin not present in other sequenced diatoms. The characterisation of a bacteria-like rhodopsin in F. cylindrus was conducted by applying reverse genetics tools. The genome was characterised by a low G+C content, which affected codon usage. High sequence polymorphism resulted in pronounced unequal expression of putative heterozygous allelic gene copies in response to six different conditions. RNA-Seq detected transcriptional activity for 95% of the 27,137 predicted genes and > 4 fold expression changes between 55% of putative alleles. The most significant transcriptional changes were detected during prolonged darkness affecting 70% of genes and 30% of RNA-Seq reads mapped to unannotated regions of the genome. Two rhodopsin alleles showed unequal bi-allelic expression in response to iron starvation and heterologous expression in Xenopus laevis oocytes experimentally confirmed light-driven proton pumping for the iron-induced rhodopsin allele, suggesting significance for the adaptation of F. cylindrus to environmental conditions of the Southern Ocean. These data show how the polar environment can shape the genome of a eukaryotic phytoplankton in unprecedented detail. High numbers of species-specific genes resulting in expansion of gene and protein families, low G+C likely enabling efficient translation at low temperatures and a high degree of heterozygosity combined with unequal bi-allelic expression, may provide an adaptive strategy to polar conditions by conferring metabolic flexibility and capacity to adapt to a rapidly changing environment